Field of the Invention
The present invention relates to methods for producing an aqueous suspension of precious metal nanoparticles, in particular, to methods for producing an aqueous suspension of gold nanoparticles for bioconjugation to functional ligands including bio-molecules.
Description of the Related Art
Precious metal nanoparticles (PMNPs) and colloidal PMNPs, also called precious metal nanocolloids (PMNCs), are being widely investigated for their potential use in a wide variety of biological and medical applications. Applications of the PMNCs include using the PMNC as an imaging agent, a sensing agent, a gene-regulating agent, a targeted drug delivery carrier, or as a photoresponsive antibacterial therapeutic agent. Most of these applications require a surface modification on the PMNPs, which is also referred to as a surface functionalization.
In the past most PMNCs have been made by chemical synthesis processes such as those based on a reduction of the precious metal in an ionic state or those based on forming complex ions with ligand molecules. Inherently, chemical syntheses produce chemical by-products such as those which are formed as a result of the counterpart reaction during the reduction of the precious metals resulting in residual ions in an electrolyte of the colloidal solution. Furthermore, currently commercially-available PMNCs made by chemical syntheses contain stabilizing agents that prevent the PMNPs from aggregating and precipitating out of the colloidal solution. The presence of the stabilizing agents or residual ions of the chemical by-products could cause instability of a colloidal system during a subsequent bioconjugation process. However, it is desirable for the surface functionalization of PMNP for bio-applications to yield stable resultant PMNCs without precipitation of the nanoparticles.
FIG. 1A is a flowchart showing a chemical synthesis process in the prior art for generation of nanoparticles followed by bioconjugation. As shown, process 100 starts with a colloidal solution of PMNPs (i.e., PMNCs) generated by a chemical synthesis method at step 102. After fabrication of the PMNCs, the colloidal solution is stored in a container, such as a capped glass container, for a period of time (step 104) until the subsequent bioconjugation process (step 106). The preparation step 102 may be done by purchasing commercially-available PMNCs, which are usually delivered and stored in a container.
Ligand exchange reactions have been found to be a powerful approach for surface modification of various inorganic colloidal nanoparticles including the PMNCs and are used to produce organic and water-soluble nanoparticles with various core materials and functional groups. One of the most difficult aspects of applying the ligand exchange reactions to the PMNCs is to achieve substantially complete ligand exchange as well as to preserve the stability of the colloidal suspension during the reaction.
Pulsed laser ablation in liquid (PLAL) is a method suitable for synthesizing functional nanoparticles directly from bulk materials, and can provide totally ligand-free nanoparticles. Commonly owned U.S. Patent Application Pub. No. 2012/0225021 discloses a method of producing stable bare colloidal gold nanoparticles in water by a top-down fabrication method using a PLAL method, with bulk gold as a target material. The results demonstrated colloidal stability of gold nanoparticles during surface functionalization with thiolated polyethylene glycol (PEG) characterized by monitoring the change of the absorbance of the localized surface plasmon resonance of gold nanocolloids at 520 nanometers (nm).
FIG. 1B is a flowchart showing a PLAL method in the prior art for generation of nanoparticles for bioconjugation. As shown, at step 112, a precious metal (PM) target material and a suspension liquid are provided for process 101. At step 114, PMNPs are generated by focusing laser pulses on the PM target material. The generated PMNPs are combined with the suspension liquid to form a colloidal suspension that has the PMNPs. At step 116, the colloidal suspension is stored in a container for a period of time (i.e., a storage period), and then used in a bioconjugation process to combine a bio-molecule ligand with the PMNP (step 118).
Notwithstanding such recent advancements in PLAL methods, when the overall process is considered from the generation of the PMNPs to the bioconjugation reaction at least two challenges remain. One is accurate size control of the PMNPs in the nanoparticle generation process. Another is ion concentration control of electrolytes during a subsequent procedure for conditioning the produced PMNCs specifically for bioconjugation reactions.
C. Rehbock et al. (Phys. Chem. Chem. Phys., “Size control of laser-fabricated surfactant-free gold nanoparticles with highly diluted electrolytes and their subsequent bioconjugation”, published on 3 Oct. 2012, DOI: 10.1039/C2CP42641B) demonstrated a nanoparticle size control process. The embodiment described generation of gold nanoparticles (AuNPs) for bioconjugation by using a nanosecond PLAL approach and a size control process with a highly diluted electrolyte. More specifically, the AuNPs are generated and dispersed into a carrier steam of water containing a trace amount of salts. To control the size of the AuNPs generated by the PLAL method with the highly diluted electrolyte, C. Rehbock et al. demonstrated a possibility of size control of the AuNPs by introducing a known amount of specific ions into the water. To produce the AuNPs in a diameter of 10 nm or larger, C. Rehbock et al. shows that a precise control of ion concentration is required, because the produced size of AuNPs changes strongly depending on the ion concentration when the ion concentration is in a range below 30 micromole (μM). At such low concentration ranges the effect of a trace amount of externally introduced ions, such as a contamination, on nanoparticle size are no longer negligible.
There are various ways to analyze individual ions in the electrolyte based on an element analysis such as inductively coupled plasma mass spectroscopy (ICP-MS), or based on molecular analyses such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), Fourier transform infrared spectroscopy (FTIR) and Raman scattering (RS). However, all of these measurements are too costly and time consuming to perform every time before bioconjugation in order to evaluate the capability of the PMNCs for bioconjugation.
Thus, it is desirable to predict the capability of the PMNCs for bioconjugation and to control ion concentrations of the electrolytes of the solutions that the PMNCs are in.